Direct in vitro histone homocysteinylation by HTL. a Schematic illustration of protein modification by HTL. b Dot-blot analysis of histone homocysteinylation by HTL. The unmodified histones H3, H4, H2a, and H2b expressed from E. coli were used. Top panel: four histones were incubated with 5 mM HTL for 2 h and histone homocysteinylation was detected using anti-Hcy antibodies. ( + : positive control, tubulin antibody diluted 1:1000 was used as the positive control; – : negative control, sodium phosphate buffer was used as the negative control); Middle panel: histone H3 was treated with 5 mM HTL for 2, 6, and 14 h, respectively, and histone homocysteinylation was detected using anti-Hcy antibodies. Bottom panel: histone H3 was treated with 0.5 mM, 1 mM, 5 mM, and 10 mM HTL respectively for 2 h and histone homocysteinylation was detected using anti-Hcy antibodies. c MALDI analysis of unmodified H3 from E. coli with (bottom) or without (top) in vitro HTL treatment. The undigested H3 display a major peak of about 15KD. Additional major peaks greater than 15KD are seen in HTL-treated H3 samples. The difference in molecular mass between the adjacent two peaks is in the proximity of 3 Hcy modifications, indicating that multiple, simultaneous KHcy modifications may exist on H3 during HTL treatment. The x and y axes represent m / z and relative ion intensity, respectively. d A typical HPLC-MS/MS spectra of a tryptic peptide 'GVLK Hcy VFLENVIR' derived from HTL-treated H4 with homocystylation at H4K59 site. The x and y axes represent m / z and relative ion intensity, respectively. A series of b- and y-type homocysteinylation fragment ions are evident which not only provide reliable sequence information, but also indicate an unambiguous +174.04600 Da shift for Hcy. e Illustration of histone homocysteinylation sites identified by HPLC-MS/MS analysis on unmodified core histones treated with HTL. The green diamond shape depicts homocysteinylation sites in core histones (H3, H4, H2a, and H2b). The number underneath each red lysine residue (K) represents the position of the particular lysine residue within each respective histone. Homocysteinylation sites, present both naturally in normal human brain samples (Fig. 1b ) and after in vitro HTL treatment are marked with a red dot
{}